Biochemometry identifies suppressors of pro-inflammatory gene expression in Pterocarpus santalinus heartwood

The heartwood extract of the Ayurvedic medicinal plant Pterocarpus santalinus L. f. has previously been shown to significantly suppress the expression of CX3CL1 and other pro-inflammatory molecules in IL-1-stimulated human endothelial cells. Here, we identify the pigment-depleted extract PSD as the most promising yet still complex source of metabolites acting as an inhibitor of CX3CL1 gene expression. For the target-oriented identification of the constituents contributing to the observed in vitro anti-inflammatory effect of PSD, the biochemometric approach ELINA (Eliciting Nature's Activities) was applied. ELINA relies on the deconvolution of complex mixtures by generating microfractions with quantitative variances of constituents over several consecutive fractions. Therefore, PSD was separated into 35 microfractions by means of flash chromatography. Their 1H NMR data and bioactivity data were correlated by heterocovariance analysis. Complemented by LC-MS-ELSD data, ELINA differentiated between constituents with positive and detrimental effects towards activity and allowed for the prioritization of compounds to be isolated in the early steps of phytochemical investigation. A hyphenated high-performance counter-current chromatographic device (HPCCC+) was employed for efficient and targeted isolation of bioactive constituents. A total of 15 metabolites were isolated, including four previously unreported constituents and nine that have never been described before from red sandalwood. Nine isolates were probed for their inhibitory effects on CX3CL1 gene expression, of which four isoflavonoids, namely pterosonin A (1), santal (6), 7,3'-dimethylorobol (12) and the previously unreported compound pterosantalin A (2), were identified as pronounced inhibitors of CX3CL1 gene expression in vitro.